A crucial element in the development of effective prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is a relevant model since it induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS.&#8232;&#8232;Our studies have focused on the use of the highly attenuated modified vaccinia virus Ankara to express SIV proteins, including env, and/or gag-pol. Immunization with MVA-SIV recombinants resulted in a reduction in setpoint plasma viral load that was associated with prolonged survival. Vaccination was associated with better preservation of memory CD4+ T cells that in turn correlated with titers of neutralizing antibodies to the challenge virus prior to challenge suggesting an important role for NAb and Envelope immunogens in this model.&#8232;The purpose of our current vaccine studies has been to use a prime-boost strategy to generate antibody that will neutralize the pathogenic SIVsmE660 and study the role of this antibody in the SIV model. Similar studies using SIVmac239 have been hampered due to the neutralization resistance associated with its envelope glycoprotein.&#8232;&#8232;We generated a MVA recombinant that expresses the less neutralization-resistant SIVsmE543-3 envelope and a plasmid of the codon-optimized gp140 (also SIVsmE543-3) that we used to generate recombinant trimeric envelope. A cohort of 24 rhesus macaques (6 per group) were immunized with either: A) 543 env trimer + AbISCO-100 adjuvant alone, B) MVA-nonrecombinant + adjuvant, C) Two MVA-Env primes, + two trimer boosts with adjuvant, and D) MVA-Env plus trimer co-administered 4 times with adjuvant. All SIV Env immunized animals developed neutralizing antibodies to SIVsmE660 but only weak and partial neutralization of SIVsmE543. They were challenged intrarectally with a single dose of SIVsmE660, 4 months following the last immunization. A delay and blunting of viremia was observed in 50% of animals that received the prime boost regimen with one animal not apparently infected (Group C). However a macaque each from Groups B and D also remained uninfected. All uninfected animals were rechallenged with the same IR dose of SIVsmE660. We are currently evaluating vaccine-induced cellular immune responses to determine immune correlates of the partical protection observed. Future studies will focus on the prime/boost protocol with challenge at peak immune responses (4 weeks after last immunization) using a repetitive, lower dose SIVsmE660 challenge model. &#8232;&#8232; We collaborated with a number of groups on vaccine studies using SIVsmE543 immunogens or SIVsmE660 challenge viruses including: 1) Dr. Barbara Felber and George Pavlakis at the NCI, 2) Dr. Harriet Robinson at Emory and 3) Dr. Dan Barouch (Harvard) and Nelson Michael (WRAIR). In this latter study, MVA and adenovirus expressing SIVsmE543-3 gag-pol and env were combined as immunogens. This strategy led of a delay in acquisition of infection by the heterologous SIVmac251 despite the fact that high titers of tier 2 neutralizing antibodies were not generated by this strategy. DEVELOPMENT OF ALTERNATIVE CHALLENGE STRAINS&#8232;The development and characterization of alternative to the major SIVmac239/2251 and SIVsmE660 challenge strains are required for studies evaluating the role of neutralizing antibody in protection. We wish to develop and characterize additional strains of SIV that are more sensitive to neutralization and that can model the Tier2 type sensitivity of primary HIV isolates. We are evaluating two full length infectious clones of SIVsmE660. These both replicate efficiently in macaque PBMC in vivo, are sensitive to neutralization and replicate efficiently in rhesus macaques in vivo. As described in the pathogenesis project (AI000370) these clones are still sensitive to TRIM restriction and recent studies from the Watkins and Letvin labs suggest that TRIM5 polymorphisms and in particular expression of the restrictive TRIM-TFP allele contributes to protection in low dose SIVsmE660 challenge models. Therefore we have generated minimal point mutants in CA that are now resistant to TRIM restriction that will be evaluated in vivo. These clones would be ideal source of challenge strains that should avoid TRIM restriction as a confounding factor in low dose E660 challenge models.In addition, we have generated clones with a more neutralization resistant phenotype that is intermediate between the original E660 clones and 543-3 that may better model the phenotype of primary HIV-1 strains.